Cyclin-dependent protein kinases (CDK) are same cell cycle regulation of key signaling molecules. Hartwell, Nurse and Hunt awarded the 2001 Nobel Prize for revealing CDK cell cycle control mechanisms. Uncontrolled cell cycle is one of the hallmarks of cancer, and CDK over activity leads to cell proliferation. Therefore, in recent years, the focus of the research and development of targeted anti-cancer drugs in the world has begun to shift to CDK inhibitors.
CDK4/6 inhibitor had been listed as Palbociclib of Pfizer (Ibrance, 2015), Ribociclib of Novartis (Kisqali, 2017) and Abemaciclib of Iilly (Verzenio, 2017). However, due to the weak anticancer effectives of the CDK4/6 inhibitors, need combined with other anticancer drugs such as Letrozole, and with same serious adverse reactions and drug resistance.
CDK1 is the only one that must be used in cell proliferation and has become the latest target of cancer drug research. [Nature, 2007, 448: 811-816] Therefore, CDK1 is targeted of the research and development of anti-cancer drugs. Flavonoids are same natural CDK1 selective inhibitors, widely exist in many fruits, vegetables and herbs, with many kinds of biological activity as anti-thrombotic, anti-inflammatory, immunity to normal, antiviral, anti-cancer, and so on. Flavonoids are not directly kill the cells, but based on the regulation of intracellular reactive oxygen species (ROS), thus selectively induce apoptosis, inhibit proliferative and metastasis of solid cancers by target activating intracellular apoptosis pathway and bypassing extrinsic death receptor pathway, and protecting normal cells. [Eur. J. Med. Chem., 2012, 49: 24-40]
There are different mechanisms of oxidation and reduction within normal cells and tumor cells. Proliferation and metastasis of solid cancers cells need a lot of oxygen. So, hypoxia is an important characteristic of solid cancers microenvironment. In normal cells, in the state of oxygen enrichment, a small amount of .O2− is capturing by superoxide dismutase (SOD), with releasing H2O2, and decomposing by glutathione (GPX) and catalase (CAT). A small amount of 0.02″ is capturing too by polyphenolic compounds (as flavonoids) similarly SOD, with releasing H2O2, and decomposing by GPX and CAT. In cancer cells by lacking SOD, GPX and CAT, in the lack of oxygen enrichment, a large amount of .O2− is collecting with promoting the proliferation of cancer cells. A large amount of .O2− is capturing by flavonoids with releasing and collecting H2O2, which high concentration of H2O2 can specific target oxidative the enzymes with active site of cysteine (such as CDC25, HIF-1α, caspase), thus activating mitochondria apoptosis pathway without the death receptor pathway, thereby, selectively inhibit proliferation and induce apoptosis of cancer cells. [Biomedicine & Pharmacotherapy, 2005, 59: 169-174; Cancer Letters, 2007, 252: 1-8]
However, due to flavonoids with poor solubility and the strong “liver first pass effect” and “hepatoenteral circulation” of glucose aldehyde acid metabolism (>95%) by oral, so that bioavailability of flavonoids is very low. Therefore, to improve druggability, based on the structure modification of flavonoids have an important practical value. [Eur. J. Pharmacology, 2010, 630: 121-130] The structure modifications of flavonoids by organic amines are most remarkable.

In the structure modifications of flavonoids with general organic amine (no Mannich bases) substituent, as Flavopiridol, P276-00 and Voruciclib, due to do not format the stable six-member ring structure by intermolecular hydrogen bond between phenol hydroxyl and amine substituent, the “liver firs effect” by oral and “hepatoenteral circulation” cannot be effective inhibited with little bioavailability. Meanwhile, the flavonoids nucleus is not activated by substituent bases with druggability little excellent.
Scutellaria baicalensis is herb drug from Chinese with main active Baicalin, Baicalein as hydrolysis product of Baicalin is stronger anti-proliferation activity than Baicalin. [Cancer Treatment Reviews, 2009, 35: 57-68] Baicalein is an inhibitor of CDK1 with selectively inducing apoptosis of cancer cells with little toxic effect on normal cells by regulating ROS. [Eur J Pharmacology, 2010, 630: 121-130; Neuroscience Letters, 2008, 444: 264-269] So, Baicalein as a lead compound for structural modification is a great significance. The Mannich derivatives of Baicalein, a preparation method and thereof with the inhibition activity of protein kinase C, was public on CN1427003A. 8-aminemethyl-Baicalein Mannich derivatives, a preparation method and thereof with the inhibition activity of CDK1, was public on CN200910140275.4 and U.S. Pat. No. 8,377,895B2. 8-(4-hydroxy-piperidin-1-ylmethyl)-2-phenyl-chromen-4-one (BA j) is the best CDK1 selective inhibitor, and different from the cytotoxic drugs with damaging cancer cells also normal cells. By using the differential mechanisms of oxidation and reduction within normal cells, cancers cells and activated lymphocyte, BA-j can target activate intrinsic apoptosis pathway and pass extrinsic death receptor pathway, thus selectively induce apoptosis, inhibit proliferative of cancer cells and activated lymphocyte, and protect normal cells. The druggability of BA-j is excellent in the application. [Scientific Reports, 2015, 5: 13626; Anti-Cancer Agents in Medicinal Chemistry, 2016, 16: 914-924; Fitoterapia, 2015, 107: 36-34; RSC Advances, 2015, 5: 89818-89826; Bioorganic & Medicinal Chemistry, 2008, 16: 7127-7132]
In structure modifications of BA-j by Mannich bases substituent, due to format the stable six-member ring structure by intermolecular hydrogen bond between phenolic hydroxyl and Mannich amine, the “liver firs pass effect” of glucose aldehyde acid metabolism (<5%) by oral and “hepatoenteral circulation” are effective inhibited, and bioavailability significantly improved. Due to log P of BA-j is lower, high protein binding rate and strong affinity, mainly distributed in the blood, so the blood system exceptions (such as metastasis cancer cell into blood, leukemia and AIDS) good effect of elimination of symptoms caused by abnormal immune.
Chrysin is also a natural flavonoid with extensive pharmacological activities, is one of the major components of propolis, and has the anticancer and radiotherapy sensitization, inhibit the aromatase activity, anti-inflammatory, anti-oxidation, the prevention and treatment of disease of heart head blood-vessel and other pharmacological effects. The inhibition of VEGF by HIF-1α can inhibit the formation of new blood vessels and selectively induce apoptosis of cancer cells and inhibit the metastasis of solid cancer [Molecular Cancer Therapeutics, 2007, 6: 220-226] Therefore, it is of great significance to modify the structure of Chrysin in order to obtain a new type of anti-solid cancers drug with high efficiency and low toxicity.
Flavopiridol is an organic amine derivative of Chrysin as the first generic CDKs inhibitor with significantly improving the sensitivity of chemotherapy drugs, at the same time also found that can prevent HIV-1 in cell proliferation, show the good prospects of cancer and AIDS. The subsequent application of chemotherapy can effectively improve the sensitivity of chemotherapy drugs and bring new ideas and hope to the treatment of cancer and AIDS. However, it is a pan-CDKs inhibitor. The solubility is poor, and the intravenous drip steady plasma concentration low (0.27 μM), by oral which resulted in a stronger “liver first pass effect” with low bioavailability. It is not easy to be degraded into tissue cells with producing strong cytotoxic effects and causing severe secretion of diarrhea and other adverse reactions. The druggability of Flavopiridol is not excellent. [Blood, 2009, 113: 2637-2644; Life Sciences, 1998, 62: 1861-1873; Invest New Drugs, 2012, 30: 629-638]
P276-00 is an organic amine derivative of Chrysin as a selective inhibitor of CDK 4, 1, 9 with the anticancer activity and drug potency superior to that of Flavopiridol, which had little effect on normal pulmonary fibroblasts, as wt-38 and MRC-5, and had entered phase III clinical stage. There is little diarrhea and other adverse reactions with comparing Flavopiridol. There is the similar druggability problem of Flavopiridol. [Molecular Cancer Therapeutics, 2007, 6: 918-925, 918-925; Leukemia, 2009, 23: 961-970]
Vorciclib of similar P276-00 is another organic amine derivative of Chrysin as a selective inhibitor of CDK 4, 1, 9. There are more than 30 types of cancer cell lines that show a more anticancer activity, and currently being treated with other drugs for melanoma. There is the similar druggability problem of P276-00 and Flavopiridol.
The substituent of Mannich base derivative of Chrysin on the existing technology had 6-aminemethyl, 8-aminemethyl, and 6,8-diaminemethyl [Synlett, 2006, (8): 1225-1229; Shenyang Pharmaceutical University J. , 2010, 27: 448-452; Nature Product Communications, 2011, 6: 31-34; Ukrainica Bioorganica Acta2, 2013, 3-7; Anti-Cancer Agents in Medicinal Chemistry, 2016, 16: 914-924] The anticancer activity of the Mannich base derivatives of Chrysin had a little improvement.